Vitamin A is required for a variety of biological processes. The studies described within the body of the grant proposal are directed at understanding how cellular retinol binding proteins function in retinol uptake, metabolism, and transport. It is our hypothesis that these proteins are more than just passive reservoirs which prevent non-specific partitioning of the hydrophobic vitamin into cellular membranes: The proteins themselves are involved in protein-protein interactions with the enzymes which metabolize retinoids, and as a consequence regulate retinol metabolism and function. With site-directed mutagenesis and a variety of techniques we will examine the following aspects of cellular retinol binding protein (CRBP) and cellular retinol binding protein type II(CRBP(II)): (1) How the intracellular transport proteins for vitamin A deliver retinol to the metabolizing enzymes. This is a process which requires protein-protein recognition. We will determine the sites on the carrier proteins involved in this recognition. (2) How the ligand enters the binding cavities of CRBP and CRBP(II). The X-ray structures are available for both proteins and it is apparent that a conformational change must accompany ligand entry and exit. We describe experiments aimed at determining the nature of this conformational change. (3) CRBP discriminates between retinol and retinal, whereas CRBP(II) does not. We will determine the structural basis for this functional difference. (4) The role of water in ligand binding. The binding cavities of CRBP and CRBP(II) contain specifically bound water molecules in addition to the ligand retinol. We postulate that there is a functional purpose, in addition to the obvious structural purpose, for the inclusion of water molecules in the binding site.